RESUMO
CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma (DLBCL) patient-derived xenograft (PDX) model. Whereas the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy, rather CD70 interaction in cis with the nanoCAR induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9, resulted in dramatically enhanced functionality in the DLBCL PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knock out (KO) CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that WT CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. On the other hand, the gene signature of KO CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products that led to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.
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Type I interferon (IFN) is a potent antitumoral drug, with an important history in the treatment of hematologic malignancies. However, its pleiotropic nature leads to severe dose-limiting toxicities that blunt its therapeutic potential. To achieve selective targeting of specific immune or tumor cells, AcTakines (Activity-on-Target Cytokines), i.e., immunocytokines utilizing attenuated cytokines, and clinically optimized A-Kines™ were developed. In syngeneic murine models, the CD20-targeted murine IFNα2-based AcTaferons (AFNs) have demonstrated clear antitumoral effects, with excellent tolerability. The current study explores the antitumoral potential of the humanized huCD20-Fc-AFN in 5 different humanized patient derived xenograft (PDX) models of huCD20+ aggressive B non-Hodgkin lymphomas (B-NHLs). The huCD20-Fc-AFN consists of a huCD20-specific single-domain antibody (VHH) linked through a heterodimeric 'knob-in-hole' human IgG1 Fc molecule to an attenuated huIFNα2 sequence. An in vitro targeting efficacy of up to 1.000-fold could be obtained, without detectable in vivo toxicities, except for selective (on-target) and reversible B cell depletion. Treatment with huCD20-Fc-AFN significantly increased the median overall survival (mOS) in both non-humanized (mOS 31 to 45 days; HR = 0.26; p = 0.001), and humanized NSG/NOG mice (mOS 34 to 80 days; HR = 0.37; p < 0.0001). In humanized mice, there was a trend for increased survival when compared to equimolar rituximab (mOS 49 to 80 days; HR = 0.73; p = 0.09). The antitumoral effects of huCD20-Fc-AFN were partly due to direct effects of type I IFN on the tumor cells, but additional effects via the human immune system are essential to obtain long-term remissions. To conclude, huCD20-Fc-AFN could provide a novel therapeutic strategy for huCD20-expressing aggressive B-NHLs.
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Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 have complementary roles in angiogenesis and promote an immunosuppressive tumor microenvironment. It is anticipated that the combination of VEGF and ANG2 blockade could provide superior activity to the blockade of either pathway alone and that the addition of VEGF/ANG2 inhibition to an anti-programmed cell death protein-1 (PD-1) antibody could change the tumor microenvironment to support T-cell-mediated tumor cytotoxicity. Here, we describe the pharmacologic and antitumor activity of BI 836880, a humanized bispecific nanobody comprising two single-variable domains blocking VEGF and ANG2, and an additional module for half-life extension in vivo. BI 836880 demonstrated high affinity and selectivity for human VEGF-A and ANG2, resulting in inhibition of the downstream signaling of VEGF/ANG2 and a decrease in endothelial cell proliferation and survival. In vivo, BI 836880 exhibited significant antitumor activity in all patient-derived xenograft models tested, showing significantly greater tumor growth inhibition (TGI) than bevacizumab (VEGF inhibition) and AMG386 (ANG1/2 inhibition) in a range of models. In a Lewis lung carcinoma syngeneic tumor model, the combination of PD-1 inhibition with VEGF inhibition showed superior efficacy versus the blockade of either pathway alone. TGI was further increased with the addition of ANG2 inhibition to VEGF/PD-1 blockade. VEGF/ANG2 inhibition had a strong antiangiogenic effect. Our data suggest that the blockade of VEGF and ANG2 with BI 836880 may offer improved antitumor activity versus the blockade of either pathway alone and that combining VEGF/ANG2 inhibition with PD-1 blockade can further enhance antitumor effects. SIGNIFICANCE STATEMENT: Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 play key roles in angiogenesis and have an immunosuppressive effect in the tumor microenvironment. This study shows that BI 836880, a bispecific nanobody targeting VEGF and ANG2, demonstrates substantial antitumor activity in preclinical models. Combining VEGF/ANG2 inhibition with the blockade of the PD-1 pathway can further improve antitumor activity.
Assuntos
Neoplasias , Fator A de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiopoietina-2/metabolismo , Receptor de Morte Celular Programada 1 , Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Inibidores da Angiogênese , Neoplasias/tratamento farmacológico , Morte Celular , Angiopoietina-1 , Microambiente TumoralRESUMO
ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Antígenos Virais/imunologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/química , Antígenos Virais/genética , Camelídeos Americanos , Linhagem Celular Tumoral , Células Epiteliais/virologia , Epitopos/química , Epitopos/imunologia , Humanos , Evasão da Resposta Imune/genética , Soros Imunes/química , Modelos Moleculares , Mutação , Testes de Neutralização , Ligação Proteica , Estrutura Secundária de Proteína , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/isolamento & purificação , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.
Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Técnicas de Química Combinatória/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Citotóxicas Formadoras de Poros/imunologia , Anticorpos de Domínio Único/imunologia , Potência de Vacina , Animais , Morte Celular , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Pneumonia/imunologia , Pneumonia/microbiologia , Pneumonia/patologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologiaRESUMO
Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.
Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Proteínas Virais de Fusão/antagonistas & inibidores , Administração por Inalação , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antivirais/imunologia , Antivirais/metabolismo , Antivirais/farmacologia , Feminino , Expressão Gênica , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/virologia , Masculino , Modelos Moleculares , Cavidade Nasal/efeitos dos fármacos , Cavidade Nasal/imunologia , Cavidade Nasal/virologia , Testes de Neutralização , Palivizumab/biossíntese , Palivizumab/imunologia , Palivizumab/farmacologia , Pichia/genética , Pichia/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/patogenicidade , Sigmodontinae , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
In 1989, a new type of antibody was identified, first in the sera of dromedaries and later also in all other species of the Camelidae family. These antibodies do not contain a light chain and also lack the first constant heavy domain. Today it is still unclear what the evolutionary advantage of such heavy chain-only antibodies could be. In sharp contrast, the broad applicability of the isolated variable antigen-binding domains (VHH) was rapidly recognized, especially for the development of therapeutic proteins, called Nanobodies(®). Here we summarize first some of the unique characteristics and features of VHHs. These will next be described in the context of different experimental therapeutic applications of Nanobodies against different viruses: HIV, Hepatitis B virus, influenza virus, Respiratory Syncytial virus, Rabies virus, FMDV, Poliovirus, Rotavirus, and PERVs. Next, the diagnostic application of VHHs (Vaccinia virus, Marburg virus and plant Tulip virus X), as well as an industrial application (lytic lactococcal 936 phage) will be described. In addition, the described data show that monovalent Nanobodies can possess unique characteristics not observed with conventional antibodies. The straightforward formatting into bivalent, multivalent, and/or multispecific Nanobodies allowed tailoring molecules for potency and cross-reactivity against viral targets with high sequence diversity.
Assuntos
Anticorpos Antivirais/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Região Variável de Imunoglobulina/uso terapêutico , Viroses/diagnóstico , Viroses/tratamento farmacológico , Vírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Camelídeos Americanos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Viroses/imunologiaRESUMO
Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection.
Assuntos
Anticorpos Neutralizantes/sangue , Hepacivirus/imunologia , Hepatite C/terapia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Modelos Animais de Doenças , Genótipo , Hepacivirus/genética , Pan troglodytes , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genéticaRESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis worldwide. Viral attachment and entry, representing the first steps of virus-host cell interactions, are major targets of adaptive host cell defenses. The mechanisms of antibody-mediated neutralization by host neutralizing responses in HCV infection are only poorly understood. Retroviral HCV pseudotypes (HCVpp) and recombinant cell culture-derived HCV (HCVcc) have been successfully used to study viral entry and antibody-mediated neutralization. METHODS: In this study, we used these model systems to investigate the mechanism of antibody-mediated neutralization by monoclonal antienvelope antibodies and polyclonal anti-HCV immunoglobulins purified from HCV-infected patients. RESULTS: Using a panel of monoclonal antienvelope antibodies, we identified an epitope within the E1 glycoprotein targeted by human neutralizing antibodies during postbinding events. Interestingly, we observed that host neutralizing responses in the majority of HCV-infected individuals include antibodies targeting HCV entry after binding of the virus to the target cell membrane. Using a kinetic assay based on HCVpp and HCVcc entry, we demonstrate that purified antiviral immunoglobulins derived from individual HCV-infected patients appear to inhibit HCV infection at an entry step closely linked to CD81 and scavenger receptor BI (SR-BI). CONCLUSIONS: Our results indicate that host neutralizing responses in HCV-infected patients target viral entry after HCV binding most likely related to HCV-CD81, and HCV-SR-BI interactions, as well as membrane fusion. These findings have implications not only for the understanding of the pathogenesis of HCV infection but also for the design of novel immunotherapeutic and preventive strategies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica/tratamento farmacológico , Fusão de Membrana/efeitos dos fármacos , Receptores Depuradores Classe B/imunologia , Adulto , Idoso , Anticorpos Anti-Idiotípicos/imunologia , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Células Cultivadas , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Receptores Virais , Receptores Depuradores Classe B/efeitos dos fármacos , Receptores Depuradores Classe B/metabolismo , Tetraspanina 28 , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.
Assuntos
Anticorpos Monoclonais/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular , Mapeamento de Epitopos , Anticorpos Anti-Hepatite C/isolamento & purificação , Humanos , Camundongos , Testes de Neutralização , Pan troglodytes , Proteínas do Envelope Viral/imunologiaRESUMO
Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2(bxd) (BALB/c x C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.
Assuntos
Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Imunoterapia/métodos , Animais , Feminino , Vacinas contra Hepatite B/uso terapêutico , Vírus da Hepatite B/imunologia , Imunização Secundária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmídeos/genética , Plasmídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vaccinia virus/genética , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003). In this study, we characterized the HCV-HS interaction and analyzed its inhibition by antiviral host immune responses. Using recombinant envelope glycoproteins, virus-like particles, and HCV pseudoparticles as model systems for the early steps of viral infection, we mapped viral and cellular determinants of HCV-HS interaction. HCV-HS binding required a specific HS structure that included N-sulfo groups and a minimum of 10 to 14 saccharide subunits. HCV envelope binding to HS was mediated by four viral epitopes overlapping the E2 hypervariable region 1 and E2-CD81 binding domains. In functional studies using HCV pseudoparticles, we demonstrate that HCV binding and entry are specifically inhibited by highly sulfated HS. Finally, HCV-HS binding was markedly inhibited by antiviral antibodies derived from HCV-infected individuals. In conclusion, our results demonstrate that binding of the viral envelope to a specific HS configuration represents an important step for the initiation of viral infection and is a target of antiviral host immune responses in vivo. Mapping of viral and cellular determinants of HCV-HS interaction sets the stage for the development of novel HS-based antiviral strategies targeting viral attachment and entry.
Assuntos
Hepacivirus/fisiologia , Hepacivirus/patogenicidade , Heparitina Sulfato/metabolismo , Proteínas do Envelope Viral/fisiologia , Animais , Anticorpos Antivirais , Antígenos Virais , Sítios de Ligação , Linhagem Celular , Mapeamento de Epitopos , Células HeLa , Hepacivirus/genética , Hepacivirus/imunologia , Heparitina Sulfato/química , Hepatite C Crônica/etiologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Camundongos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , VirulênciaRESUMO
Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Fator C1 de Célula Hospedeira/imunologia , Proteínas do Envelope Viral/metabolismo , Antígenos Virais/metabolismo , Sequência de Bases , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Regulação da Expressão Gênica , Hepacivirus/genética , Hepatite C/genética , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Dados de Sequência Molecular , Análise Serial de Proteínas , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Técnicas de Cultura de Tecidos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
BACKGROUND: There is little information on the timing, magnitude, specificity, and clinical relevance of the antibody response to acute hepatitis C virus (HCV) infection. We investigated the specificity, titer, and neutralizing potential of antibody responses to acute infection by examining 12 injection drug users before, during, and after infection. METHODS: Seroconversion was defined as incident detection of HCV-specific antibodies by using a commercially available enzyme-linked immuosorbent assay (ELISA). HCV protein-specific antibody responses were measured using recombinant antigens in an ELISA. For neutralization assays, plasma was incubated with human immunodeficiency virus (HIV)-HCV H77 or control HIV-murine leukemia virus (MLV) pseudotype virus and then allowed to infect Hep3B hepatoma cells. RESULTS: The mean time to HCV seroconversion was 6 weeks after the onset of viremia. Antibody responses to nonstructural proteins were detected before responses to the structural proteins, and antibodies to both were primarily restricted to the immunoglobulin G1 (IgG1) subclass. The maximum median end point titers for antibody responses to structural and nonstructural proteins were 1 : 600 and 1 : 6400, respectively. Antibodies that neutralized a retroviral pseudotype bearing HCV 1a envelope glycoproteins were detected at seroconversion in only 1 subject and at 6-8 months after seroconversion in 3 subjects. The delayed appearance of neutralizing antibodies was consistent with the late development of antibodies specific for the viral envelope glycoproteins, which are believed to mediate virus neutralization. CONCLUSION: The humoral immune response to acute HCV infection is of relatively low titer, is restricted primarily to the IgG1 subclass, and is delayed. A better understanding of why production of neutralizing antibody is delayed may improve efforts to prevent HCV infection.
Assuntos
Formação de Anticorpos/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C/imunologia , Doença Aguda , Adulto , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , RNA Viral/sangue , Fatores de Tempo , Proteínas Virais/imunologia , ViremiaRESUMO
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Interaction of dendritic cells (DCs) with viral particles may play an important role in the immunopathogenesis of HCV infection. Since the synthesis or purification of infectious virions is limited, we used HCV-like particles (HCV-LPs) to study the interaction of HCV with human DCs. Immature DCs exhibited an envelope-specific and saturable binding of HCV-LPs, indicating receptor-mediated DC-HCV-LP interaction. Confocal microscopy revealed that HCV-LPs were rapidly taken up by DCs in a temperature-dependent manner. Competition experiments demonstrated that C-type lectins such as mannose receptor or DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing nonintegrin) were not sufficient for mediating HCV-LP binding. HCV-LP uptake was followed by DC activation. DCs pulsed with HCV-LPs stimulated HCV core-specific CD4(+) T cells, indicating that uptake of HCV-LPs by DCs leads to antigen processing and presentation on major histocompatibility complex (MHC) class II molecules. Finally, HCV-LP-derived antigens were efficiently cross-presented to HCV core-specific CD8(+) T cells. These findings demonstrate that HCV-LPs represent a novel model system to study HCV-DC interaction allowing definition of the molecular mechanisms of HCV uptake, DC activation, and antigen presentation to T cells. Furthermore, HCV-LP-mediated DC activation and efficient antigen presentation may explain the marked immunogenicity of HCV-LPs in vivo.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Hepacivirus/imunologia , Hepatite C/etiologia , Vírion/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Hepatite C/imunologia , Antígenos de Histocompatibilidade Classe II , Humanos , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
The tolerability and immunogenicity of the hepatitis C virus E1 protein as a candidate vaccine was examined in a Phase I, single-arm study. Twenty healthy male volunteers were injected in the deltoid muscle at weeks 0, 3 and 6 with 20 microg recombinant E1 adsorbed on alum. A fourth (booster) dose was administered to 19 subjects at week 26. The candidate therapeutic vaccine was well tolerated. Three vaccine doses induced a clear humoral anti-E1 response that was boosted by a fourth dose. A strong, specific cellular immune response towards E1 was elicited in all vaccine recipients, which included a clear Th1 type response in all but one of the subjects.
Assuntos
Hepatite C/imunologia , Hepatite C/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Adolescente , Adulto , Formação de Anticorpos/imunologia , Anticorpos Anti-Hepatite C/análise , Anticorpos Anti-Hepatite C/biossíntese , Humanos , Imunidade Celular/imunologia , Imunização Secundária , Injeções Intramusculares , Interferon gama/análise , Interferon gama/biossíntese , Interleucina-5/análise , Interleucina-5/biossíntese , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/efeitos adversos , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversosRESUMO
Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.
Assuntos
Hepacivirus/imunologia , Hepacivirus/fisiologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Hepatite C/imunologia , Hepatite C/virologia , Doença Aguda , Linhagem Celular Tumoral , Mapeamento de Epitopos , Hepacivirus/classificação , Humanos , Soros Imunes/imunologia , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vírion/classificação , Vírion/imunologia , Vírion/metabolismoRESUMO
Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.
Assuntos
Moléculas de Adesão Celular/fisiologia , Hepacivirus/fisiologia , Lectinas Tipo C/fisiologia , Lisossomos/metabolismo , Receptores de Superfície Celular/fisiologia , Células Cultivadas , Células Dendríticas/fisiologia , Endossomos/virologia , Células Endoteliais/virologia , Humanos , Proteínas do Envelope Viral/metabolismo , Vírion/fisiologiaRESUMO
New treatments are needed for chronic hepatitis C patients in whom viral clearance cannot be achieved. Thirty-five chronic hepatitis C patients (genotype 1) were randomized to receive 20 mug of recombinant HCV E1 (E1) (n = 26) or placebo (n = 9) intramuscularly at weeks 0, 4, 8, 12, and 24. Thirty-four then received open-label E1 vaccine at weeks 50, 53, 56, 59, 62, and 65. Twenty-four patients (12 men, 12 women; mean age, 52 y; 18 interferon-based treatment failures; mean baseline alanine aminotransferase [ALT] level, 118 IU/L) underwent a biopsy before and after 2 courses of E1, 17 months later. Liver histology was scored by 2 blinded pathologists according to the Ishak and Metavir systems. Postinjection reactions were similar to placebo (alum only). Nine of 24 patients (38%) had improvement of 2 points or more, 10 (41%) remained stable, and 5 (21%) showed worsening in total Ishak score. Nine patients (38%) improved both on Ishak and Metavir fibrosis scores. Plasma HCV-RNA levels remained unchanged, whereas ALT levels showed a trend toward a decrease during treatment. All but 3 patients developed a significant de novo E1-specific T-cell response. The increase in anti-E1 antibody levels correlated with the decrease in total Ishak score and with the relative decreases in both Ishak fibrosis score and ALT level (all P < or =.01). In conclusion, E1 therapeutic vaccination is well tolerated and the observed effects warrant further study.
Assuntos
Hepatite C Crônica/tratamento farmacológico , Proteínas Estruturais Virais/uso terapêutico , Alanina Transaminase/sangue , Formação de Anticorpos , Feminino , Fibrose , Hepacivirus/genética , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , RNA Viral/análise , Proteínas Estruturais Virais/efeitos adversos , Proteínas Estruturais Virais/imunologiaRESUMO
The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.